© 2000 by European Society of Cardiology
Copyright © 2000, European Society of Cardiology
Involvement of protein kinase C in superoxide anion-induced activation of nuclear factor-
B in human endothelial cells
aDepartment of Cardiovascular Medicine, Kumamoto University School of Medicine, 2-2-1 Honjo, Kumamoto 860-0811, Japan
bDepartment of Pharmacology, Kumamoto University School of Medicine, 2-2-1 Honjo, Kumamoto 860-0811, Japan
* Corresponding author. Tel.: +81-96-373-5076; fax: +81-96-373-5078 hideyuki{at}gpo.kumamoto-u.ac.jp
Received 14 April 1999; accepted 11 October 1999
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Abstract |
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 Top Abstract 1 Introduction2 Methods3 Results4 DiscussionAcknowledgmentsReferences |
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Objective: Nuclear factor-kappa B (NF-
B) plays an important role in the regulation of redox-sensitive genes which are related to the pathogenesis of various vascular diseases. Although oxygen free-radicals are known to activate NF-B, the signaling pathway of oxygen free radical-induced NF-B activation remains largely unclear. Thus, this study was performed to examine the possible involvement of protein kinase C (PKC) in the oxygen free radical-induced NF-B activation in human umbilical vein endothelial cells (HUVECs). Methods: Superoxide anion was generated by xanthine and xanthine oxidase. An electrophoretic mobility shift assay (EMSA) was performed using a B-motif oligonucleotide and nuclear extracts from HUVECs. Immunoblot analysis using an antibody against IB
, phosphorylated by IB kinase, or myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylated by protein kinase C was carried out. An NF-B luciferase reporter gene assay was also performed. Results: The treatment of the cells with superoxide anion for 60 min increased the NF-B/DNA binding activity. Immunoblot analysis showed that superoxide anion induced phosphorylation of IB within 10 min. Furthermore, phosphorylation of MARCKS occurred more rapidly than phosphorylation of IB. Pretreatment of the cells with calphostin C (100–400 nmol/l) and chelerythrine chloride (5–10 µmol/l), inhibitors of PKC, abolished the superoxide anion-induced NF-B activation. Down-regulation of endogenous PKC by long-term exposure to phorbol 12-myristate 13-acetate decreased the superoxide anion-induced NF-B activation to a basal level. Superoxide anion induced the luciferase reporter gene and this induction was completely inhibited by calphostin C (200 nmol/l) and 4,5-dihydroxy-1,3-benzene disulfonic acid (tiron). Conclusion: These results suggest that PKC is involved in the activation of NF-B by superoxide anion in human endothelial cells.
KEYWORDS Atherosclerosis; Endothelial function; Free radicals; Protein kinases; Signal transduction
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionAcknowledgmentsReferences |
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Oxidative stress has been reported to play important roles in the pathogenesis of various cardiovascular diseases including atherosclerosis [1–4]. One of the major effects of oxidative stress in cardiovascular diseases is the induction of proinflammatory and proatherothrombogenic molecules, such as vascular cell adhesion molecule-1 (VCAM-1) or tissue factor (TF) in vascular cells. These molecules are believed to be immediate early genes (IEGs) and their expressions have been reported to be increased by oxygen free-radicals. Most of these redox-sensitive genes have single or multiple
B-motifs in the promoter or enhancer region, which bind to the transcription factor NF-B, a pleiotropic transcription factor [5–8]. Oxygen free-radicals have been known to activate NF-B in a variety of cells [2,9]. Activation of NF-B requires phosphorylation of serine residues at 32 and 36 (Ser-32 and Ser-36, respectively) in IB by IB kinase and subsequent degradation through a common ubiquitin–proteasome pathway [10,11]. However, the signaling pathway of the oxygen free radicals-induced NF-B activation remains largely unclear.
Protein kinase C (PKC) has been reported to be an activator of IB kinase, because phorbol 12-myristate 13-acetate (PMA), a potent activator of PKC, induced the phosphorylation of IB followed by strong activation of NF-B in a variety of cells [9,12]. We previously reported that lysophosphatidylcholine (lysoPC), one of the active components of oxidized low density lipoprotein, activates NF-B in cultured human endothelial cells [13]. Experiments with PKC inhibitors suggested that the PKC-mediated pathway was involved in this activation. Recently, we showed that PKC was activated in human atherosclerotic coronary arteries with the specific antibody against myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylated by PKC (anti-P-MARCKS antibody) [14]. From these results, we consider the possibility that the oxygen free-radicals in the human vascular wall may activate PKC, which may in turn activate NF-B via activation of IB kinase in endothelial cells. The present study was designed to examine the involvement of PKC in the oxidative stress-induced activation of NF-B in cultured human endothelial cells.
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2 Methods |
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2.1 Cell culture
Primary cultures of human umbilical vein endothelial cells (HUVECs) were obtained by collagenase digestion and were incubated in medium 199 with 15% fetal calf serum (FCS), endothelium growth supplement (30 mg/l), heparin (100 mg/l), penicillin (100 000 Unit/l) and streptomycin (100 mg/l) at 37°C in a humidified atmosphere of 95% air/5% CO2 as described [13]. Confluent cultures of HUVECs exhibited the typical cobblestone morphology, and most of these cells contained factor VIII-related antigen, determined using indirect immunofluorescence as described [13].
2.2 Experimental protocols
After reaching confluence in 100-mm plastic dishes, HUVECs were rinsed with serum-free medium 199 and further cultured in serum-free medium 199 for 4 h before the experiments. HUVECs were rinsed with serum-free medium 199 and then treated in serum-free medium 199 with xanthine (100 µmol/l) and xanthine oxidase (20 Unit/l) (X/XO) or PMA (100 nmol/l) in the presence or absence of PKC inhibitors or other additives for the indicated times. After treatment, HUVECs were washed twice with PBS and immediately frozen on liquid nitrogen and stored at –80°C until use. The final concentration of DMSO in the culture medium was less than 0.1%, and control samples contained an identical amount of DMSO. For down-regulation of PKC, confluent HUVECs were treated with PMA (100 nmol/l) for 24 h before serum starvation. The activity of PKC in the cell extract was measured as described [14].
2.3 Preparation of nuclear and cell extracts
Nuclear extracts of HUVECs were prepared by treatment with a modified mini-scale detergent as described [13,15]. Briefly, HUVECs cultured in 100-mm plastic dishes were harvested by scraping in 800 µl of Buffer-A (mmol/l: HEPES 10, pH 7.9; KCl 10; EDTA 0.1; EGTA 0.1; DTT 1.0; PMSF 1.0; pepstatin A 2 µg/ml; leupeptin 1 µg/ml) at 4°C and kept on ice for 15 min, followed by the addition of 50 µl of 10% Nonidet P-40 (NP-40), and endothelial cells were homogenized by vigorous stirring by a vortex for 10 s. The homogenate was centrifuged at 3000xg at 4°C for 10 min to prepare endothelial nuclei, and the isolated nuclei in the pellets were resuspended in 10 µl of ice-cold Buffer-B (mmol/l: HEPES 20, pH 7.9; NaCl 400; EDTA 1.0; EGTA 1.0; DTT 1.0; PMSF 1.0; pepstatin A 2 µg/ml). Endothelial nuclear proteins were extracted by incubation of the nuclei on ice for 30 min, and then the supernatant containing the nuclear proteins was collected after centrifugation at 8000xg at 4°C for 10 min. The obtained nuclear protein preparation was stored at –80°C until use. Protein concentration was determined by the method of Bradford using bovine serum albumin as a standard. Cell extracts of HUVECs used for immunoblot analysis were prepared as previously described [14,17]. Briefly, HUVECs cultured in 100-mm plastic dishes were scraped off in 600 µl of RIPA buffer (mmol/l: Tris–HCl 60, pH 7.5; NaCl 180; EDTA 6.0; sodium pyrophosphate 18; NaF 60; DTT 1.0; PMSF 1.0; 1.2% NP-40; pepstatin A 8.3 µg/ml; leupeptin 7.5 µg/ml) at 4°C. The suspension was sonicated and centrifuged at 13 000xg at 4°C for 20 min to obtain the cell extract. The cell extract was stored at –80°C until use. Protein concentration was determined using the BCA protein assay reagent with bovine serum albumin as a standard.
2.4 Electrophoretic mobility shift assay (EMSA)
EMSA was performed as previously described [13]. Double-stranded synthetic oligonucleotides were labeled at the 5' end with [
-32P]ATP and T4 polynucleotide kinase to use as hot probes. The sequences of the probes were as follows: NF-B, 5'-CCAGAGGGGACTTTCCGAGAGG-3' (a kind gift from Dr M. Takiguchi, Department of Molecular Genetics, Kumamoto University School of Medicine); NF-B mutant, 5'-AGTTGAGGCGACTTTCCCAGGG-3' (Santa-Cruz Biotechnology, Inc. CA); Oct-1, 5'-TGTCGAATGCAAATCACTAGAA-3' (Santa-Cruz Biotechnology, Inc. CA). The nuclear protein/DNA binding reaction was performed by incubation of the nuclear protein (5 µg) and 1 fmol probe (about 2x103 cpm) in the reaction mixture on ice for 30 min. After electrophoresis in 5% acrylamide, the gel was dried and exposed onto an imaging plate for analysis of the radioactivity in the NF-B/DNA binding bands of using a Bio-Imaging analyzer FLA-2000 (Fuji Co.).
2.5 SDS-PAGE and immunoblot analysis
SDS-PAGE was performed using the method of Laemmli [16]. Immunoblotting after SDS-PAGE was performed using 125I-protein A as described [17,18]. The whole cell extract was separated in 9.0% acrylamide for PKC and MARCKS and 12.0% for IB and phospho-IB, respectively, and transferred to Immobilon-P membranes (Millipore). The membranes were incubated overnight with rabbit polyclonal antibodies to human IB (1:200; New England Biolabs, Beverly, MA), phospho-IB (1:200; New England Biolabs, Beverly, MA), MARCKS (anti-human MARCKS antibody) (1:500; Upstate Biotechnology, NY) or phosphorylated MARCKS (anti-P-MARCKS antibody) (1:100 or 1:200). The anti-P-MARCKS antibody was developed and purified as previously reported [14].
2.6 Transfection of the luciferase reporter gene and dual-luciferase reporter gene assay
The NF-B luciferase reporter gene (pNF-B-Luc) was obtained from Stratagene (La Jolla, CA). The firefly-luciferase reporter gene which has five-tandem repeats of the B-motif (T
GC x5 repeats) upstream of the TATA-box was transfected into HUVECs. To adjust the variation of transfection efficacy among culture dishes, HUVECs were co-transfected with pRL-TK vector (Promega, Co. WI) which contained the Herpes simplex virus thymidine kinase promoter to provide constitutive expression of Renilla luciferase expression. HUVECs were plated in 0.2% gelatin-coated 60-mm plastic dishes, and cultured overnight. The cells were rinsed twice with serum-free OPTI-MEM-I (Life Technologies, Inc.) and co-transfected with pNF-B-Luc (6.0 µg of plasmid DNA) and pRL-TK (0.6 µg of plasmid DNA) using 12 µl of TransIT-LT2 (Pan Vera, Co. WI) in 4 ml of serum-free OPTI-MEM-I medium for 4 h. Then, the medium was exchanged for a complete growth medium which contained medium 199, with 15% fetal calf serum (FCS), endothelium growth supplement (30 mg/l), heparin (100 mg/l), penicillin (100 000 Unit/l) and streptomycin (100 mg/l), and the cells were further cultured for 36–42 h. The cells were treated for 4 h with X/XO or PMA with or without various inhibitors. The luciferase assay was carried out with the dual-luciferase reporter assay system (Promega, Co. WI) according to the manufacturer's protocols with a luminometer (Aloka, Co.). The ratio of luminescence signals from the reaction mediated by firefly luciferase to luminescence signals from the reaction mediated by Renilla luciferase was determined.
2.7 Measurement of intracellular oxidative stress
HUVECs cultured in 60-mm plastic dishes were rinsed twice with HBSS and treated with X/XO or a vehicle for 5 min. Subsequently, 2',7'-dichlorofluorescin diacetate (DCFH-DA) (Lambda, Austria) (5 µmol/l) was loaded for 5 min in the dark. Fluorescent emission signals were visualized with a confocal laser-scanning light microscope (CLSM) (Olympus, Japan).
2.8 Materials
The following chemicals and reagents were obtained from the indicated sources: medium 199 from Life Technologies, FCS from Atlanta Biologicals, endothelium growth supplement from Becton Dickinson, xanthine, xanthine oxidase, PMA, DMSO, EDTA, EGTA, polyethylene glycol-superoxide dismutase (PEG-SOD) and 4,5-dihydroxy-1,3-benzene disulfonic acid (tiron) from Sigma; calphostin C, chelerythrine chloride and NP-40 from Calbiochem Novabiochem; poly(dI-dC) from Pharmacia Biotech; T4-polynucleotide kinase from Takara, Japan; antibodies against p50, p65, c-Rel and Rel B from Santa-Cruz Biotechnology; antibodies against human IB and phospho-IB from New England Biolabs; BCA protein assay reagent from Pierce Co.; [-32P]ATP and 125I-protein A from Amersham and NEN, respectively. Other chemicals used were of analytical grade.
2.9 Statistics
Data are expressed as means±S.E. values. Statistical evaluation of the data was performed by analysis of variance (ANOVA), when more than two groups were compared. Values were considered to be statistically significant at P<0.05.
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3 Results |
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3.1 Effects of superoxide anion on NF-
B/DNA binding activity in HUVECsEMSA showed that HUVECs endogenously contained a small amount of active NF-
B in the nucleus without any stimulants, and treatment with superoxide anion, PMA and 150 µmol/l of hydrogen peroxide (data not shown) increased the NF-B/DNA binding activity (Fig. 1A). The addition of a 1000-fold excess of unlabeled NF-B probe as a competitor (lane 4) and the use of an NF-B mutant probe (NF-Bm) (lanes 5 and 6) eliminated the bands of NF-B/DNA binding from the cells treated with or without superoxide anion (Fig. 1A). The use of BSA instead of the nuclear protein preparation eliminated the bands of protein/DNA binding (data not shown). The band for Oct-1/DNA binding was unchanged by treatment of the cells with superoxide anion (lanes 7 and 8) (Fig. 1A). The amount of activated NF-B was quantified using a Bio-Imaging analyzer (Fig. 1B). Treatment of the cells with superoxide anion and PMA increased the activity of NF-B to 185.5±18.6% (n=5, P<0.01) and 493.5±29.5% (n=5, P<0.001) of control, respectively (Fig. 1B). Pretreatment of HUVECs with PEG-SOD for 15 min did not affect the superoxide anion-induced NF-B activation (data not shown). Pretreatment of HUVECs with 10 mmol/l of tiron, a cell-permeable superoxide anion scavenger, abolished the superoxide anion-induced NF-B activation, but treatment of the cells with tiron alone did not affect the basal NF-B activity (data not shown). To determine the subunits of the superoxide anion-induced NF-B/DNA complex, we performed a supershift assay using the specific antibodies to p50, p65, c-Rel, and Rel B (Fig. 1C). The antibody to p50 caused the supershift of the NF-B/DNA complex, while the antibody to p65 blocked the NF-B/DNA binding activity. In contrast, the NF-B/DNA complex did not react with the antibodies to c-Rel and Rel B. These results indicated that the superoxide anion-induced NF-B/DNA complex included p50 and p65. These results are consistent with the finding that both p50 and p65 are involved in the formation of the NF-B/DNA complex in murine macrophage cell lines treated with hydrogen peroxide [20].
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3.2 Time course of phosphorylation of I
B in HUVECsIt is well known that phosphoryltion of I
B precedes the activation of NF-B. Therefore, we examined whether or not superoxide anion increased phosphorylation of IB (Fig. 2A). Immunoblot analysis using the antibody to phospho-IB clearly demonstrated that superoxide anion rapidly induced phosphorylation of IB within 10 min, diminishing within 60 min. This decrease in phospho-IB was thought to be due to the dephosphorylation or the degradation by an ubiquitin–proteasome pathway. When the antibody to IB was used for immunoblot analysis, no significant changes in the immunoreactivities were observed by treatment with superoxide anion (Fig. 2A). These results suggest that a relatively small amount of IB was phosphorylated and degraded by the treatment with superoxide anion. Fig. 2B summarizes the results from three independent experiments. Immunobolt analysis using the antibody to phospho-IB showed that pretreatment of the cells with calphostin C (100 nmol/l) for 15 min completely abolished the superoxide anion-induced phosphorylation of IB (data not shown).
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3.3 Time course of the activation of PKC in HUVECs
MARCKS, originally reported as a substrate only for PKC, is now known to be a good substrate for MAP kinase and cyclin-dependent protein kinases [17,18]. Since Ser-152 and Ser-156 in MARCKS are phosphorylated only by PKC, the increase in phosphorylation of these sites can be used as an indicator for PKC activation in intact cells [20]. In a previous study, we produced and purified a specific antibody (anti-P-MARCKS antibody) against MARCKS phosphorylated by PKC at Ser-152 and Ser-156 [14]. Using this antibody, we examined whether or not superoxide anion activated PKC in HUVECs (Fig. 3A). When the cells were treated with superoxide anion, the rapid and strong phosphorylation of MARCKS was observed. Fig. 3B summarizes the results from three independent experiments. Phosphorylation of MARCKS increased within 5 min and showed a peak after 10–15 min. When the antibody to MARCKS was used for immunoblot analysis, no significant changes in immunoreactivities were observed (Fig. 3A). The phosphorylation of MARCKS also increased with PMA (100 nmol/l for 5 min) as previously reported (data not shown) [14].
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3.4 Effects of calphostin C and chelerythrine chloride on the superoxide anion-induced NF-
B activation in HUVECsAs the next step, we examined whether or not the PKC-mediated signaling pathway was involved in the superoxide anion-induced NF-
B activation in HUVECs. HUVECs were pretreated with calphostin C for 15 min prior to stimulation (Fig. 4A). Treatment of the cells with calphostin C alone (400 nmol/l) did not affect the basal NF-B/DNA binding activity. Calphostin C significantly inhibited the superoxide anion-induced NF-B activation in a concentration-dependent manner. In order to further confirm the involvement of PKC in the superoxide anion-induced NF-B activation, HUVECs were pretreated with chelerythrine chloride for 15 min prior to stimulation (Fig. 4B). Chelerythrine chloride is a specific inhibitor for all isotypes of PKC by interaction with the catalytic domain via a competing phosphate acceptor. Chelerythrine chloride inhibited the activation of NF-B in a concentration-dependent manner. When 10 µmol/l of chelerythrine chloride was used, NF-B activation decreased to less than the basal level. These results suggest that a significant amount of active PKC activated NF-B even under the basal conditions. Fig. 4C summarizes the results of statistical analysis. Treatment of the cells with superoxide anion significantly increased the NF-B activity to 176.8±10.6% (n=5). Pretreatment of the cells with 200 and 400 nmol/l of calphostin C significantly decreased NF-B activity to119.8±4.1 and 86.7±3.8% (n=5), respectively. Pretreatment of the cells with 5 and 10 µmol/l of chelerythrine chloride significantly decreased NF-B activity to 78.5±4.4 and 38.5±4.2% (n=5), respectively.
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3.5 Effects of the down-regulation of PKC by long-term treatment with PMA on the superoxide anion-induced NF-
B activation in HUVECsTo further confirm the involvement of PKC in the superoxide anion-induced NF-
B activation, PKC in HUVECs was down-regulated by pretreatment with 100 nmol/l of PMA for 24 h. Treatment of HUVECs with 100 nmol/l of PMA for 24 h decreased PKC activity to a basal level (data not shown). As shown in Fig. 5A, down-regulation of PKC significantly inhibited both PMA- and superoxide anion-induced NF-B activation in HUVECs. Fig. 5B summarizes statistical analysis of the results from five independent experiments. No significant increase in NF-B activity was observed for the samples of nuclear extracts from the cells treated with superoxide anion or PMA after down-regulation of PKC.
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3.6 Effects of calphostin C and tiron on the superoxide anion-induced NF-
B activationWe wondered whether or not the NF-
B activation by superoxide anion would increase the NF-B-mediated transcription in HUVECs. For this purpose, we performed an NF-B dual-luciferase reporter gene assay (Fig. 6). Treatment of the cells with PMA and superoxide anion increased the luciferase activities to 495.0 and 260.0% of the control value, respectively, in triplicate determinations. Pretreatment of the cells with tiron abolished the superoxide anion-induced increase in luciferase activity to a basal level. To our knowledge, this is the first report on the stimulative effect of superoxide anion on the transcriptional activity using the NF-B luciferase reporter gene assay system. Furthermore, the increase in luciferase activity with superoxide anion was inhibited by pretreatment of the cells with calphostin C in a concentration-dependent manner (Fig. 6). These results further supported our idea that PKC is involved in the superoxide anion-induced activation of NF-B.
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3.7 Effects of the extracellular superoxide anion on the intracellular oxidative stress
Furthermore, we asked whether or not the extracellular superoxide anion could increase the intracellular oxidative stress in HUVECs. The intracellular oxidative stress was assessed by measurement of the changes in fluorescence resulting from the oxidation of an intracellular probe [19]. DCFH-DA enters the cells and is subsequently cleaved by the cellular erastases, followed by the trapping of the non-fluorescent 2',7'-dichlorofluorescin (DCFH) inside. The oxidation of DCFH by oxygen-free radicals, particularly hydrogen peroxide, makes a fluorescent product, DCF. Treatment of the cells with superoxide anion rapidly increased the DCFH oxidation in HUVECs (Fig. 7). These results suggested that the extracellular superoxide anion increased the intracellular oxidative stress in HUVECs.
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4 Discussion |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionAcknowledgmentsReferences |
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In the present study, we examined the possibility that activation of PKC was involved in the superoxide anion-induced NF-
B activation in HUVECs. The two different types of inhibitors of PKC, as well as down-regulation of PKC, completely abolished the superoxide anion-induced NF-B activation. These results strongly suggested that activation of PKC was necessary for the superoxide anion-induced NF-B activation. Using the anti-P-MARCKS antibody, we demonstrated that superoxide anion rapidly activated PKC in HUVECs. Since phosphorylation of MARCKS was faster than that of IB, it is intriguing to speculate that PKC activated with the superoxide anion activated IB kinase. This is the first report to show the involvement of PKC in the oxidative stress-induced NF-B activation in human endothelial cells. Kaul et al. [21] reported that PKC was not involved in the hydrogen peroxide-induced NF-B activation in murine macrophage cell lines. The reason for this discrepancy between the studies is not clear at present, but may be due to the different mechanisms of the NF-B activation between HUVECs and macrophages. The activation of PKC by treatment with superoxide anion was detected, because phosphorylation of MARCKS at Ser-152 and Ser-156 strongly increased by the treatment. However, it remains undetermined from the present study which isotypes of PKC are involved in the superoxide anion-induced NF-B activation. Calphostin C is a competitive inhibitor for the diacyl glycerol and phorbol ester-binding site in the regulatory domain of both conventional PKC (cPKC) and novel PKC (nPKC) [12]. Since both conventional PKC (cPKC) and novel PKC (nPKC) have been reportedly down-regulated by long-term treatment with PMA [22], the effects of calphostin C and down-regulation of PKC on the NF-B activation suggested that some isotypes of cPKC and nPKC were responsible for the superoxide anion-induced NF-B activation. In the preliminary experiments, HUVECs contained the and isotypes in cPKC, and the 
and 
isotypes in nPKC. These isotypes were translocated from the cytosol fraction to the membrane fraction by treatment with PMA (data not shown). However, no significant translocation of these isotypes (, , and ) was observed by treatment with superoxide anion (data not shown). Other unknown isotypes of PKC, or unknown mechanisms of PKC activation may participate in the superoxide anion-induced PKC activation. In addition to translocation, tyrosine phosphorylation of PKC has been reported to be another indicator of its activation in some signal transduction systems. Konishi et al. [23] reported that hydrogen peroxide induced constitutive, phospholipid-independent activation of PKC in COS-7 cells. This activation was due to the phosphorylation of PKC at tyrosine residues in the catalytic domain [23]. We examined the tyrosine phosphorylation of PKC after treatment with superoxide anion by immunoblot analysis with an anti-phosphotyrosine antibody following the immunoprecipitation of each isotype. However, we could not obtain the clear evidence of phosphorylation at tyrosine residues in any isotype of PKC in HUVECs (data not shown).
In addition, we found using a DCFH probe that the extracellular superoxide anion could increase the intracellular oxidative stress. Since the oxidation of DCFH is mainly caused by hydrogen peroxide, but not by superoxide anion, superoxide anion could be converted to hydrogen peroxide by superoxide dismutase (SOD) immediately after it entered the cells. Pretreatment of the cells with the cell-permeable PEG-SOD did not affect the superoxide anion-induced NF-B activation by EMSA. These results suggested that HUVECs contained a high amount of SOD sufficient to convert superoxide anion into hydrogen peroxide and that hydrogen peroxide was an active principle in the cells.
Immunoblot analysis showed that superoxide anion induced phosphorylation of IB, but no clear change was seen in the levels of IB itself. Since phosphorylation of IB and the resultant activation of NF-B did occur, it was highly likely that degradation of IB occurred by treatment of the cells with superoxide anion. The amount of degradation of IB seemed to be too small to be detectable by immunoblot analysis in the present study.
We previously reported that lysoPC caused the burst production of superoxide anion [24], and that lysoPC activated NF-B [13]. Together with these results, the present results suggest that the reactive oxygen species produced endogenously by lysoPC in HUVECs were the active principles in the lysoPC-induced NF-B activation. We also reported that the activation of NF-B by lysoPC was suppressed by calphostin C and chelerythrine chloride [13].
The activation of PKC by PMA has been reported to induce endogenous production of reactive oxygen species through NAD(P)H oxidase in a variety of cells. Therefore, we examined the production of superoxide anion by PMA in HUVECs using the lucigenin-elicited chemiluminescence method. We found that a much lower amount of superoxide anion was produced by PMA (100–1000 nmol/l) than lysoPC (7.5 µmol/l) and that tiron did not inhibit the PMA-induced NF-B activation by EMSA (data not shown). These results suggested that superoxide anion produced by PMA did not play an important role in the PMA-induced NF-B activation under our experimental conditions.
In a recent clinical investigation, the long-term supplementation with -tocopherol was found to prevent vascular events and improve endothelial functions in patients with atherosclerotic coronary arteries [25]. Alpha-tocopherol was also found to inhibit the activity of PKC in a variety of cells [26,27]. Therefore, -tocopherol may prevent the oxidant stress-induced endothelial activation, not only by quenching oxygen free radicals produced in the atherosclerotic arterial walls, but also by inhibiting the oxidative stress-induced PKC activation.
Time for primary review 26 days.
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Acknowledgments |
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We gratefully acknowledge Dr K. Matsui (Fukuda Hospital) and Dr K. Fukunaga (Kumamoto University) for technical support. This work was supported in part by the Grants-in-Aid for Scientific Research and for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture, and the Smoking Research Foundation, Tokyo, Japan.
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